Production of chemicals from micro-organisms has been an important application of biotechnology. Typically, the steps in developing such a bio-production method may include 1) selection of a proper micro-organism host, 2) elimination of metabolic pathways leading to by-products, 3) deregulation of desired pathways at both enzyme activity level and the transcriptional level, and 4) overexpression of appropriate enzymes in the desired pathways. In preferred aspect, the present invention has employed combinations of the steps above to redirect carbon flow from phenylalanine or tyrosine through enzymes of the plant phenylpropanoid pathway which supplies the necessary precursor for the desired biosynthesis of resveratrol.
Resveratrol (or 3,4,5-trihydroxystilbene) is a phytophenol belonging to the group of stilbene phytoalexins, which are low-molecular-mass secondary metabolites that constitute the active defense mechanism in plants in response to infections or other stress-related events. Stilbene phytoalexins contain the stilbene skeleton (trans-1,2-diphenylethylene) as their common basic structure: that may be supplemented by addition of other groups as well (Hart and Shrimpton, 1979, Hart, 1981). Stilbenes have been found in certain trees (angio-sperms, gymnosperms), but also in some herbaceous plants (in species of the Myrtaceae, Vitaceae and Leguminosae families). Said compounds are toxic to pests, especially to fungi, bacteria and insects. Only few plants have the ability to synthesize stilbenes, or to produce them in an amount that provides them sufficient resistance to pests.
The synthesis of the basic stilbene skeleton is pursued by stilbene synthases. So far, two enzymes have been designated as a stilbene synthase; pinosylvine synthase and resveratrol synthase. To date, the groundnut (Arachis hypogaea) resveratrol synthase has been characterised in most detail, such that most of the properties are known (Schoppner and Kindl, 1984). Substrates that are used by stilbene synthases are malonyl-CoA, cinnamoyl-CoA or coumaroyl-CoA. These substances occur in every plant because they are used in the biosynthesis of other important plant constituents as well such as flavonoids, flower pigments and lipids.
Resveratrol (FIG. 1 trans-form) consists of two closely connected phenol rings and belongs therefore to the polyphenols. While present in other plants, such as eucalyptus, spruce, and lily, and in other foods such as mulberries and peanuts, resveratrol's most abundant natural sources are Vitis vinifera, -labrusca, and -muscadine (rotundifolia) grapes, which are used to make wines. The compound occurs in the vines, roots, seeds, and stalks, but its highest concentration is in the skin (Celotti et al., 1996), which contains 50-100 μg/g. (Jang et al. 1997). During red wine vinification the grape skins are included in the must, in contrast to white wine vinification, and therefore resveratrol is found in small quantities in red wine only. Resveratrol has, besides its antifungal properties, been recognized for its cardioprotective- and cancer chemopreventive activities; it acts as a phytoestrogen, an inhibitor of platelet aggregation (Kopp et al, 1998; Gehm et al 1997; Lobo et al 1995), and an antioxidant (Jang et al., 1997; Huang 1997). These properties explain the so-called French Paradox, i.e. the wine-drinking French have a low incidence of coronary heart disease despite a low-exercise, high-fat diet. Recently it has been shown that resveratrol can also activate the SIR2 gene in yeast and the analogous human gene SIRT1, which both play a key role in extending life span. Ever since, attention is very much focused on the life-span extending properties of resveratrol (Hall, 2003, Couzin, 2004). American health associations, such as the Life Extension Foundation, are promoting the vast beneficial effects of this drug, and thereby propelling the ideal conditions for a successful commercialisation. Present production processes rely mostly upon extraction of resveratrol, either from the skin of grape berries, or from Knot weed. This is a labour intensive process and generates low yield which, therefore, prompts an incentive for the development of novel, more efficient and high-yielding production processes.
In plants, the phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolic compounds, including lignins, salicylates, coumarins, hydroxycinnamic amides, pigments, flavonoids and phytoalexins. Indeed formation of resveratrol in plants proceeds through the phenylpropanoid pathway. The amino acid L-phenylalanine is converted into trans-cinnamic acid through the non-oxidative deamination by L-phenylalanine ammonia lyase (PAL) (FIG. 2). Next, trans-cinnamic acid is hydroxylated at the para-position to 4-coumaric acid (4-hydroxycinnamic acid) by cinnamate-4-hydroxylase (C4H), a cytochrome P450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450 reductase (CPR). The 4-coumaric acid, is subsequently activated to 4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). Finally, resveratrol synthase (VST) catalyses the condensation of a phenylpropane unit of 4-coumaroyl-CoA with malonyl CoA, resulting in formation of resveratrol.
Recently, a yeast was disclosed that could produce resveratrol from 4-coumaric acid that is found in small quantities in grape must (Becker et al. 2003). The production of 4-coumaroyl-CoA, and concomitant resveratrol, in laboratory strains of S. cerevisiae, was achieved by co-expressing a heterologous coenzyme-A ligase gene, from hybrid poplar, together with the grapevine resveratrol synthase gene (vst1). The other substrate for resveratrol synthase, malonyl-CoA, is already endogenously produced in yeast and is involved in de novo fatty-acid biosynthesis. The study showed that cells of S. cerevisiae could produce minute amounts of resveratrol, either in the free form or in the glucoside-bound form, when cultured in synthetic media that was supplemented with 4-coumaric acid.
However, said yeast would not be suitable for a commercial application because it suffers from low resveratrol yield, and requires addition of 4-coumaric acid, which is only present in few industrial media. In order to facilitate and broaden the application of resveratrol as both a pharmaceutical and neutraceutical, it is therefore highly desirable to obtain a yeast that can produce resveratrol directly from glucose, without addition of 4-coumaric acid.
A recent study (Ro and Douglas, 2004) describes the reconstitution of the entry point of the phenylpropanoid pathway in S. cerevisiae by introducing PAL, C4H and CPR from Poplar. The purpose was to evaluate whether multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. By feeding the recombinant yeast with [3H]-phenylalanine it was found that the majority of metabolized [3H]-phenylalanine was incorporated into 4-[3H]-coumaric acid, and that phenylalanine metabolism was highly reduced by inhibiting C4H activity. Moreover, PAL-alone expressers metabolized very little phenylalanine into cinnamic acid. When feeding [3H]-phenylalanine and [14C]-trans-cinnamic acid simultaneously to the triple expressers, no evidence was found for channeling of the endogenously synthesized [3H]-trans-cinnamic acid into 4-coumaric acid. Therefore, efficient carbon flux from phenylalanine to 4-coumaric acid via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and sheer biochemical coupling of PAL and C4H seems to be sufficient to drive carbon flux into the phenylpropanoid pathway. In yet another study (Hwang et al., 2003) production of plant-specific flavanones by Escherichia coli was achieved through expression of an artificial gene cluster that contained three genes of a phenyl propanoid pathway of various heterologous origins; PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor, and chalcone synthase (CHS) from the licorice plant Glycyrrhiza echinata. These pathways bypassed C4H, because the bacterial 4CL enzyme ligated coenzyme A to both trans-cinnamic acid and 4-coumaric acid. In addition, the PAL from Rhodotorula rubra uses both phenylalanine and tyrosine as the substrates. Therefore, E. coli cells containing the gene clusters and grown on glucose, produced small amounts of two flavanones, pinocembrin (0.29 g/l) from phenylalanine and naringenin (0.17 g/l) from tyrosine. In addition, large amounts of their precursors, 4-coumaric acid and trans-cinnamic acid (0.47 and 1.23 mg/liter respectively), were accumulated. Moreover, the yields of these compounds could be increased by addition of phenylalanine and tyrosine.
Whereas the enzyme from dicotylic plants utilizes only phenylalanine efficiently, several studies indicated that PAL from monocotylic plants, and some micro-organisms, utilizes tyrosine as well (Rösler et al., 1997). In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL, FIG. 3). Conversion of tyrosine by TAL results in the direct formation of 4-coumaric acid without the intermediacy of C4H and CPR. Mostly both activities reside on the same polypeptide and have very similar catalytic efficiencies, in spite of large differences in Km and turnover number. However, most PAL/TAL enzymes from plants prefer phenylalanine rather than tyrosine. The level of TAL activity is mostly lower than PAL activity, but the magnitude of this difference varies over a wide range. For example, the parsley enzyme has a Km for phenylalanine of 15-25 μM and for tyrosine 2.0-8.0 mM with turnover numbers 22 s−1 and 0.3 s−1 respectively. In contrast, the maize enzyme has a Km for phenylalanine only 15-fold higher than for tyrosine, and turnover numbers about 10-fold higher. Moreover, in the red yeasts, Rhodotorula glutinis (Rhodosporidium toruloides) and -rubra, the TAL catalytic activity is close to the PAL catalytic activity with a ratio of TAL/PAL of approximately 0.58. It is believed that the PAL enzyme in these yeasts degrades phenylalanine as a catabolic function and the trans-cinnamic acid formed is converted to benzoate and other cellular materials, whereas in plants it is thought to be merely a regulatory enzyme in the biosynthesis of lignin, isoflavonoids and other phenylpropanoids.
Recently, an open reading frame was found in the bacterium Rhodobacter capsulatus that encodes a hypothetical biosynthetic tyrosine ammonia lyase (TAL) that is involved in the biosynthesis of the chromophore of the photoactive yellow protein (Kyndt et al., 2002). This was the first time that a PAL-homologous gene was found in bacteria. The TAL gene was isolated and overproduced in Escherichia coli. The Km and kcat values for the conversion of tyrosine to 4-coumaric acid were 15.6 μM and 27.7 s−1 respectively, and for conversion of L-phenylalanine to trans-cinnamic acid were 1277 μM and 15.1 s−1 respectively. As a consequence of the smaller Km and a slightly larger kcat, the enzyme shows a strong preference for tyrosine over L-phenylalanine, with a catalytic efficiency (Km/kcat) for tyrosine of approximately 150-fold larger than for phenylalanine. The kinetic studies established that tyrosine, and not L-phenylalanine, is the natural substrate of the enzyme under physiological conditions. Very recently a study described the heterologous coexpression of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, 4-coumarate-Coa-ligase and chalcone synthase, for the production of flavonoids in E. coli (Watts et al., 2004). The simultaneous expression of all four genes, however, was not successful because of a nonfunctional cinnamate-4-hydroxylase. The substitution of phenylalanine ammonia lyase and cinnamate-4-hydroxylase by a new tyrosine ammonia lyase that was cloned from Rhodobacter sphaeroides, could, however, solved the problem and led to high-level production of the flavonone naringenin. Furthermore, said tyrosine ammonia lyase from Rhodobacter sphaeroides is also used for heterologous production of 4-coumaric acid (i.e. para-hydroxycinnamic acid) in Escherichia coli (US-A-2004059103). Evenmore, further methods for development of a biocatalyst for conversion of glucose into 4-coumaric acid are described. US-A-2004023357 discloses a tyrosine ammonia lyase from the yeast Trichosporon cutaneum for the production of coumaric acid in Escherichia coli and Saccharomyces cerevisiae. US-A-2001053847 describes the incorporation of the wild type PAL from the yeast Rhodotorula glutinis into E. coli, underlining the ability of the wildtype PAL to convert tyrosine directly to 4-coumaric acid. Moreover, there is also exemplification of incorporation of the wildtype PAL from the yeast Rhodotorula glutinis, plus a plant C4H and CPR into E. coli and S. cerevisiae. Also described is the development of a biocatalyst through mutagenesis of the wild type yeast PAL Rhodotorula glutinis with enhanced TAL activity U.S. Pat. No. 6,521,748). Neither of the aforementioned patents claim the incorporation of 4CL and VST for the production of resveratrol.
Recently, evidence was shown that the filamentous fungi A. oryzae contained the enzyme chalcone synthase (CHS) that is normally involved in the biosynthesis of flavonoids, such as naringenin, in plants (Seshime et al., 2005). Indeed it was also shown that A. oryzae contained the major set of genes responsible for phenylpropanoid-flavonoid metabolism, i.e PAL, C4H and 4CL. However, there is no evidence that A. oryzae contained a stilbene synthase such as resveratrol synthase.
The present invention now provides a micro-organism having an operative metabolic pathway comprising at least one enzyme activity, said pathway producing 4-coumaric acid and producing resveratrol therefrom or an oligomeric or glycosidically-bound derivative thereof. Such a micro-organism may be naturally occurring and may be isolated by suitable screening procedures, but more preferably is genetically engineered.
Preferably, said resveratrol or derivative is produced in a reaction catalysed by an enzyme in which endogenous malonyl-CoA is a substrate, and preferably said resveratrol is produced from 4-coumaroyl-CoA.
Said resveratrol or derivative is preferably produced from 4-coumaroyl-CoA by a resveratrol synthase which is preferably expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.
Generally herein, unless the context implies otherwise, references to resveratrol include reference to oligomeric or glycosidically bound derivatives thereof, including particularly piceid.
Thus, in certain preferred embodiments, said resveratrol synthase is a resveratrol synthase (EC 2.3.1.95) from a plant belonging to the genus of Arachis, e.g. A. glabatra, A. hypogaea, a plant belonging to the genus of Rheum, e.g. R. tataricum, a plant belonging to the genus of Vitus, e.g. V. labrusca, V. riparaia, V. vinifera, or any one of the genera Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus, Cissus, Calochortus, Polygonum, Gnetum, Artocarpus, Nothofagus, Phoenix, Festuca, Carex, Veratrum, Bauhinia or Pterolobium. 
Preferably, said 4-coumaric acid is produced from trans-cinnamic acid, suitably by an enzyme in a reaction catalysed by said enzyme in which oxygen is a substrate, NADH or NADPH is a cofactor and NAD+ or NADP+ is a product.
Thus, said 4-coumaric acid may be produced from trans-cinnamic acid by a cinnamate 4-hydroxylase, which preferably is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.
In certain preferred embodiments, including those referred to in the paragraphs above, said cinnamate-4-hydroxylase is a cinnamate-4-hydroxylase (EC 1.14.13.11) from a plant or a micro-organism. The plant may belong to the genus of Arabidopsis, e.g. A. thaliana, a plant belonging to the genus of Citrus, e.g. C. sinensis, C.×paradisi, a plant belonging to the genus of Phaseolus, e.g. P. vulgaris, a plant belonging to the genus of Pinus, e.g. P. taeda, a plant belonging to the genus of Populus, e.g. P. deltoides, P. tremuloides, P. trichocarpa, a plant belonging to the genus of Solanum, e.g. S. tuberosum, a plant belonging to the genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g. Z. mays, or other plant genera e.g. Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, Vigna. The micro-organism might be a fungus belonging to the genus Aspergillus, e.g. A. oryzae. 
Preferably, said 4-coumaric acid is produced from tyrosine in a reaction catalysed by an enzyme in which ammonia is produced and suitably, said 4-coumaric acid is produced from tyrosine by a L-phenylalanine ammonia lyase or a tyrosine ammonia lyase, e.g. tyrosine ammonia lyase (EC 4.3.1.5) from yeast or bacteria. Suitably, the tyrosine ammonia lyase is from the yeast Rhodotorula rubra or from the bacterium Rhodobacter capsulatus. 
Optionally, said tyrosine ammonia lyase is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.
Alternatively, said trans-cinnamic acid may be produced from L-phenylalanine in a reaction catalysed by an enzyme in which ammonia is produced and suitably said trans-cinnamic acid is formed from L-phenylalanine by a phenylalanine ammonia lyase.
In certain preferred embodiments, said L-phenylalanine ammonia lyase is a L-phenylalanine ammonia lyase (EC 4.3.1.5) from a plant or a micro-organism. The plant may belong to the genus of Arabidopsis, e.g. A. thaliana, a plant belonging to the genus of Brassica, e.g. B. napus, B. rapa, a plant belonging to the genus of Citrus, e.g. C. reticulata, C. clementinus, C. limon, a plant belonging to the genus of Phaseolus, e.g. P. coccineus, P. vulgaris, a plant belonging to the genus of Pinus, e.g. P. banksiana, P. monticola, P. pinaster, P. sylvestris, P. taeda, a plant belonging to the genus of Populus, e.g. P. balsamifera, P. deltoides, P. Canadensis, P. kitakamiensis, P. tremuloides, a plant belonging to the genus of Solanum, e.g. S. tuberosum, a plant belonging to the genus of Prunus, e.g. P. avium, P. persica, a plant belonging to the genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g. Z. mays or other plant genera e.g. Agastache, Ananas, Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia, Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago, Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea, Petroselinum, Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Quercus, Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria, Stylosanthes, Triticum, Trifolium, Triticum, Vaccinium, Vigna, Zinnia. The micro-organism might be a fungus belonging to the genus Agaricus, e.g. A. bisporus, a fungus belonging to the genus Aspergillus, e.g. A. oryzae, A. nidulans, A. fumigatus, a fungus belonging to the genus Ustilago, e.g. U. maydis, a bacterium belonging to the genus Rhodobacter, e.g. R. capsulatus, a yeast belonging to the genus Rhodotorula, e.g. R. rubra. 
Suitably, said L-phenylalanine ammonia lyase is expressed in said micro-organism from nucleic acid coding for said enzyme which is not native to the micro-organism.
Preferably, 4-coumaroyl-CoA is formed in a reaction catalysed by an enzyme in which ATP and CoA are substrates and ADP is a product and suitably 4-coumaroyl-CoA is formed in a reaction catalysed by a 4-coumarate-CoA ligase.
Said 4-coumarate-CoA ligase may be a 4-coumarate-CoA ligase (EC 6.2.1.12) from a plant, a micro-organism or a nematode. The plant may belong to the genus of Abies, e.g. A. beshanzuensis, B. firma, B. holophylla, a plant belonging to the genus of Arabidopsis, e.g. A. thaliana, a plant belonging to the genus of Brassica, e.g. B. napus, B. rapa, B. oleracea, a plant belonging to the genus of Citrus, e.g. C. sinensis, a plant belonging to the genus of Larix, e.g. L. decidua, L. gmelinii, L. griffithiana, L. himalaica, L. kaempferi, L. laricina, L. mastersiana, L. occidentalis, L. potaninii, L. sibirica, L. speciosa, a plant belonging to the genus of Phaseolus, e.g. P. acutifolius, P. coccineus, a plant belonging to the genus of Pinus, e.g. P. armandii P. banksiana, P. pinaster, a plant belonging to the genus of Populus, e.g. P. balsamifera, P. tomentosa, P. tremuloides, a plant belonging to the genus of Solanum, e.g. S. tuberosum, a plant belonging to the genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g. Z. mays, or other plant genera e.g. Agastache, Amorpha, Cathaya, Cedrus, Crocus, Festuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Pelargonium, Petroselinum, Physcomitrella, Picea, Prunus, Pseudolarix, Pseudotsuga, Rosa, Rubus, Ryza, Saccharum, Suaeda, Thellungiella, Triticum, Tsuga. The micro-organism might be a filamentous fungi belonging to the genus Aspergillus, e.g. A. flavus, A. nidulans, A. oryzae, A. fumigatus, a filamentous fungus belonging to the genus Neurospora, e.g. N. crassa, a fungus belonging to the genus Yarrowia, e.g. Y. lipolytica, a fungus belonging to the genus of Mycosphaerella, e.g. M. graminicola, a bacterium belonging to the genus of Mycobacterium, e.g. M. bovis, M. leprae, M. tuberculosis, a bacterium belonging to the genus of Neisseria, e.g. N. meningitidis, a bacterium belonging to the genus of Streptomyces, e.g. S. coelicolor, a bacterium belonging to the genus of Rhodobacter, e.g. R. capsulatus, a nematode belonging to the genus Ancylostoma, e.g. A. ceylanicum, a nematode belonging to the genus Caenorhabditis, e.g. C. elegans, a nematode belonging to the genus Haemonchus, e.g. H. contortus, a nematode belonging to the genus Lumbricus, e.g. L. rubellus, a nematode belonging to the genus Meilodogyne, e.g. M. hapla, a nematode belonging to the genus Strongyloidus, e.g. S. rattii, S. stercoralis, a nematode belonging to the genus Pristionchus, e.g. P. pacificus. 
Optionally, a NADPH:cytochrome P450 reductase (CPR) has been recombinantly introduced into said micro-organism. This may be a plant CPR introduced into a non-plant micro-organism. Alternatively, a native NADPH:cytochrome P450 reductase (CPR) has been overexpressed in said micro-organism.
In certain preferred embodiments, including those referred to in the paragraphs above, said NADPH:cytochrome P450 reductase is a NADPH: cytochrome P450 reductase (EC 1.6.2.4) from a plant belonging to the genus of Arabidopsis, e.g. A. thaliana, a plant belonging to the genus of Citrus, e.g. C. sinensis, C.×paradisi, a plant belonging to the genus of Phaseolus, e.g. P. vulgaris, a plant belonging to the genus of Pinus, e.g. P. taeda, a plant belonging to the genus of Populus, e.g. P. deltoides, P. tremuloides, P. trichocarpa, a plant belonging to the genus of Solanum, e.g. S. tuberosum, a plant belonging to the genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g. Z. mays, or other plant genera e.g. Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, Vigna. 
Whilst the micro-organism may be naturally occurring, preferably at least one copy of at least one genetic sequence encoding a respective enzyme in said metabolic pathway has been recombinantly introduced into said micro-organism.
Additionally or alternatively to introducing coding sequences coding for a said enzyme, one may provide one or more expression signals, such as promoter sequences, not natively associated with said coding sequence in said organism. Thus, optionally, at least one copy of a genetic sequence encoding a tyrosine ammonia lyase is operatively linked to an expression signal not natively associated with said genetic sequence in said organism, and/or at least one copy of a genetic sequence encoding a L-phenylalanine ammonia lyase is operatively linked to an expression signal not natively associated with said genetic sequence in said organism.
Optionally, at least one copy of a genetic sequence encoding cinnamate 4-hydroxylase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism.
Optionally, at least one copy of a genetic sequence encoding a 4-coumarate-CoA ligase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism.
Optionally, at least one copy of a genetic sequence encoding a resveratrol synthase, whether native or not, is operatively linked to an expression signal not natively associated with said genetic sequence in said organism.
Expression signals include nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Such sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
In certain aspects the invention provides a metabolically engineered micro-organism having an operative metabolic pathway in which a first metabolite is transformed into a second metabolite in a reaction catalysed by a first enzyme, said reaction step producing ammonia, and in which said second metabolite is transformed into a third metabolite in a reaction catalysed by a second enzyme, in which oxygen is a substrate, NADPH or NADH is a cofactor and NADP+ or NAD+ is a product, and in which said third metabolite is transformed into a fourth metabolite in a reaction catalysed by a third enzyme in which ATP and CoA is a substrate, and ADP is a product, and in which said fourth metabolite is transformed into a fifth metabolite in a reaction catalysed by a fourth enzyme in which endogenous malonyl-CoA is a substrate.
The present invention also provides a metabolically engineered micro-organism having an operative metabolic pathway in which a first metabolite is transformed into a said third metabolite catalysed by a first enzyme, said reaction step producing ammonia, without the involvement of said second enzyme, and in which said third metabolite is transformed into a said fourth metabolite in a reaction catalysed by a said third enzyme in which ATP and CoA is a substrate, and ADP is a product, and in which said fourth metabolite is transformed into a said fifth metabolite in a reaction catalysed by a said fourth enzyme in which endogenous malonyl-CoA is a substrate.
The micro-organisms described above include ones containing one or more copies of an heterologous DNA sequence encoding phenylalanine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding cinnamate-4-hydroxylase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4-coumarate-CoA-ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding resveratrol synthase operatively associated with an expression signal.
They include also ones lacking cinnamate-4-hydroxylase activity, and containing one or more copies of a heterologous DNA sequence encoding tyrosine ammonia lyase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding 4-coumarate-CoA-ligase operatively associated with an expression signal, and containing one or more copies of an heterologous DNA sequence encoding resveratrol synthase operatively associated with an expression signal.
In the present context the term “micro-organism” relates to microscopic organisms, including bacteria, microscopic fungi, including yeast.
More specifically, the micro-organism may be a fungus, and more specifically a filamentous fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, or other yeast genera, e.g. Cryptococcus, Debaromyces, Hansenula, Pichia, Yarrowia, Zygosaccharomyces or Schizosaccharomyces. Concerning other micro-organisms a non-exhaustive list of suitable filamentous fungi is supplied: a species belonging to the genus Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma. 
Concerning bacteria a non-exhaustive list of suitable bacteria is given as follows: a species belonging to the genus Bacillus, a species belonging to the genus Escherichia, a species belonging to the genus Lactobacillus, a species belonging to the genus Lactococcus, a species belonging to the genus Corynebacterium, a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas, etc.
The preferred micro-organisms of the invention may be S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis or B. subtilis. 
The constructed and engineered micro-organism can be cultivated using commonly known processes, including chemostat, batch, fed-batch cultivations, etc.
Thus, the invention includes a method for producing resveratrol or an oligomeric or glycosidically-bound derivative thereof comprising contacting a non-plant cell with a carbon substrate in the substantial absence of an external source of 4-coumaric acid, said cell having the capacity to produce resveratrol or an oligomeric or glycosidically-bound derivative thereof under the conditions, in which the micro-organism may be selected from the group consisting of fungi and bacteria, especially yeast.
Said carbon substrate is optionally selected from the group of fermentable carbon substrates consisting of monosaccharides, oligosaccharides and polysaccharides, e.g. glucose, fructose, galactose, xylose, arabinose, mannose, sucrose, lactose, erythrose, threose, and/or ribose. Said carbon substrate may additionally or alternatively be selected from the group of non-fermentable carbon substrates including ethanol, acetate, glycerol, and/or lactate. Said non-fermentable carbon substrate may additionally or alternatively be selected from the group of amino acids and may be phenylalanine and/or tyrosine.
In an alternative aspect, the invention includes a method for producing resveratrol or an oligomeric or glycosidically-bound derivative thereof through heterologous expression of nucleotide sequences encoding phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase and resveratrol synthase and also a method for producing resveratrol through heterologous expression of nucleotide sequences encoding tyrosine ammonia lyase, 4-coumarate-CoA ligase and resveratrol synthase.
The invention as described above has allowed the production of yeast cells producing high levels of resveratrol. Accordingly, the invention includes a micro-organism composition comprising micro-organism cells and at least 0.4 μg/g resveratrol on a dry weight basis produced in said micro-organism cells, preferably comprising at least 0.5 μg/g of said resveratrol, more preferably at least 200 μg/g. The stated level of resveratrol can be found in the yeast cells themselves. The composition may essentially consist of said yeast cells.
The resveratrol producing microorganisms described above and the pinosylvin producing organisms described in WO2008/009728 could desirably be improved to produce higher yields by redirecting the flux through the metabolism of the microorganism.
One option is to increase the amount malonyl-CoA available for further conversion into pinosylvin and resveratrol or other stilbenoids. Increasing the amount of malonoyl-CoA will have a positive effect on the production of all stilbenes of the type given in formula I

R1, R2, R3, R4, and R5 independently are either —H or —OH because malonyl-CoA is responsible for the upper ring. The stilbene that is produced depends on the other organic acid component involved, where cinnamic acid gives pinosylvin and coumaric acid gives resveratrol. Caffeic acid will give piceatannol.
By increasing the amount of available malonyl-CoA the yield of stilbenoid can be increased. A first method involves overexpression of ACC1 to create the increased supply.
Thus, the invention further includes a recombinant micro-organism having an operative metabolic pathway in which one or more stilbenes according to the general formula I:
are formed from a precursor optionally hydroxy-substituted phenyl-2-propenoic acid or ester thereof and malonyl-CoA by the action of a stilbene synthase, wherein the amount of malonyl-CoA available for use in said pathway has been increased by providing more than a native expression level of an enzyme catalysing the reactionATP+acetyl-CoA+HCO3-=ADP+phosphate-+malonyl-CoA.
Preferably, said more than native expression level of said enzyme has been provided by replacing a native promoter of a gene expressing said enzyme with a promoter providing a higher level of expression. For instance, said native promoter is replaced with a strong constitutive yeast promoter. The strong constitutive promoter may be the promoter of one of the yeast genes TDH3, ADH1, TPI1, ACT1, GPD, TEF1, TEF2, and PGI. The promoter may optionally be native to the yeast in which stilbenoid production is to be produced.
Alternatively or additionally, said more than native expression level of said enzyme has been provided by recombinantly introducing into said micro-organism at least one exogenous genetic sequence encoding a said enzyme. This may be an acetyl coenzymeA carboxylase (ACC1-EC No. 6.4.1.2).
A second and independent strategy which also increases the yield of the stilbenes is overexpression of CPR.
Thus, the micro-organism may be recombinantly engineered to produce more than a native amount of a cytochrome P450 reductase (CPR). This may be by replacing a native promoter of a gene expressing said CPR with a promoter providing a higher level of expression, for instance with a strong constitutive yeast promoter such as the promoter of one of the yeast genes TDH3, ADH1, TPI1, ACT1 GPD, TEF1, TEF2, and PGI, which optionally may be native to the yeast itself.
The micro-organism may comprise recombinantly introduced genes expressing a phenylalanine ammonia lyase, a cinnamate 4-hydroxylase and/or a coumarate-CoA ligase or appropriate enzymes for other stilbenes.
Effect of Overexpressing ACC1
Acetyl CoenzymeA carboxylase (ACC1 EC-Number 6.4.1.2) generates malonyl-CoA according to the below reaction:
[ACC1 Reaction]ATP+acetyl-CoA+HCO3-=ADP+phosphate-+malonyl-CoA
By overexpressing ACC1 more malonyl-CoA is built up and this extra pool of malonyl-CoA is expected to generate more stilbenoids since the stilbene synthase reaction requires malonyl-CoA as building block for stilbene synthesis according to the reactions below:
[resveratrol Synthase Reaction EC-Number 2.3.1.95]3malonyl-CoA+4-coumaroyl-CoA=4 CoA+3,4′,5-trihydroxystilbene+4 CO2[Stilbene Synthase Reaction]3malonyl-CoA+cinnamoyl-CoA=4 CoA+3,5-dihydroxystilbene+4 CO2[General for any Hydroxyl Stilbene Synthase]3malonyl-CoA+hydroxycinnamoyl-CoA=4 CoA+hydroxystilbene+4 CO2
Other appropriate organic acids substituting for 4-coumaroyl-CoA produce other stilbenoids.
Effect of Overexpressing CPR
Hydroxylases, such as cinnamate 4-hyroxylase (1.14.13.11), are cytochrome P450 monooxygenases that catalyse the insertion of one atom of oxygen into an organic substrate while the other oxygen atom is reduced to water. This reaction requires NADPH according to the below reaction for the hydroxylation of cinnamic acid:trans-cinnamic acid+NADPH+H++O2=4-hydroxycinnamic acid+NADP++H2O
The active site of cytochrome P450 hydroxylases contains a heme iron center. The iron is tethered to the protein via a thiolate ligand derived from a cysteine residue. In general the mechanism is as follows:
1. The resting state of the protein is as oxidized Fe3+.
2. Binding of the substrate, cinnamic acid, initiates electron transport and oxygen binding.
3. Electrons are supplied to the p450 hydroxylase by another protein, either cytochrome P450 reductase (CPR), ferredoxins, or cytochrome b5 to reduce the heme iron.
4. Molecular oxygen is bound and split by the now reduced iron.
5. An iron-bound oxidant, oxidizes the substrate to an alcohol or an epoxide, regenerating the resting state of the p450 hydroxylase.
As described above CPR act as an electron carrier and donor for the NADPH dependent cytochrome P450 hydroxylase reaction. Thus by overexpressing CPR more electrons (NADPH) are generated for the NADPH dependent hydroxylation leading to more coumaric acid, and as a consequence more coumaric acid leads to more resveratrol by the resveratrol pathway. Similar considerations apply in the production of other stilbenoids.
Resveratrol or an oligomeric or glycosidically-bound derivative thereof or other stilbenoids so produced may be used as a nutraceutical in a dairy product or a beverage such as beer.
Resveratrol produced according to the invention may be cis-resveratrol or trans-resveratrol, but it is to be expected that the trans-form will normally predominate, as with other stilbenoids.
The invention will be further described and illustrated by the following non-limiting examples.